Photodepletion cells from GVHD patients demonstrate Treg inhibitory activity. (A) Effector (E) PBMCs from GVHD patients were exposed (filled bars) or not (clear bars) to photodepletion and cocultured at indicated ratios with target (T) PBMCs from the same patient for 5 days. Proliferation was evaluated by measuring ³H-thymidine incorporation. Bars represent mean ± SEM of 6 independent experiments. (B) Experiment similar to (A) at a 1:1 ratio, where specific proliferation of CD4⁺ cells was measured by incorporation of EdU. Shown are a representative and a compilation of 3 independent experiments (mean ± SD). (C) Untreated, photodepletion-exposed, or CD4⁺CD25⁺-depleted/photodepletion-exposed PBMCs from GVHD patients were cocultured at indicated ratios with autologous PBMCs for 5 days. Proliferation was evaluated by measuring ³H-thymidine incorporation (n = 3). (D-E) PBMCs from healthy donors were stimulated by MHC-incompatible PBMCs at a 1:1 ratio and exposed (filled) or not (clear bars) to photodepletion. Cells were either left untouched, depleted, or repleted in CD4⁺CD25⁺ cells and placed into coculture with equal numbers of MLR cells from the same donors for 5 days. (D) CD4⁺ cell proliferation was assessed by EdU incorporation (mean ± SEM), and (E) CD25⁺FOXP3⁺IL-10⁺ cells within the CD4⁺ cell population (mean ± SD) was estimated by flow cytometry. *P < .05 and ***P < .001